CN101370825B - Novel FSH glycosylation variant D3N - Google Patents
Novel FSH glycosylation variant D3N Download PDFInfo
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- CN101370825B CN101370825B CN2007800030734A CN200780003073A CN101370825B CN 101370825 B CN101370825 B CN 101370825B CN 2007800030734 A CN2007800030734 A CN 2007800030734A CN 200780003073 A CN200780003073 A CN 200780003073A CN 101370825 B CN101370825 B CN 101370825B
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- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
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Abstract
FSH mutant with increased glycosylation and longer half-life is described. The use of this FSH mutant for inducing folliculogenesis in human patients is also described.
Description
Technical field
The present invention relates to the human reproduction field.More particularly, the present invention relates to fertility treatment.
Background of invention
Gonad-stimulating hormone
Follicular stimulating hormone (FSH) is a member of gonad-stimulating hormone family, and gonad-stimulating hormone plays keying action in mankind's fertility.Gonad-stimulating hormone also comprises lutropin (LH) and chorionic-gonadotropin hormone (CG), is heterodimer, and each molecule is made up of a total α-subunit (92 amino acid) and a distinctive β-subunit (FSH is 111 amino acid).The aminoacid sequence of adult form FSH α-subunit and β-subunit is respectively shown in SEQ ID NO:1 and SEQ ID NO:2.
From hypophysis and postmenopause urine liquid, isolated people FSH (EP 322,438) and in mammalian cell recombinant production (US 5,639,640, US 5,156; 957, US 4,923, and 805, US 4,840,896, US 5; 767,251, EP 211,894 and EP 521,586).People FSH β-subunit gene is also disclosed in latter's document.US 5,405, and 945 disclose a kind of modification people α-subunit gene that contains an intron.
People such as Liu, J Biol Chem 1993,15; 268 (2): 21613-7, people such as Grossmann, Mol Endocrinol199610 (6): 769-79, Roth and Dias (Mol Cell Endocrinol 1,995 1; 109 (2): 143-9, people such as Valove, Endocrinology 1994; 135 (6): 2657-61, people such as Yoo, JBiol Chem 199325; 268 (18): 13034-42), US 5,508; 261 with people such as Chappel, 1998, Human Reproduction; Disclose FSH various structures-functional relationship research at 13 (3): 1835, and identified the amino-acid residue of participating in receptors bind and activation and dimerisation.
The application of gonad-stimulating hormone in auxiliary procreation technology
Gonad-stimulating hormone plays keying action in the reproductive cycle; In exogenous treatment, use gonad-stimulating hormone also to be absolutely necessary for auxiliary procreation technology (ART); Sperm injection (IVF/ICSI) and embryo transfer (ET) and ovulation induction (OI) in for example vitro fertilization of these auxiliary procreation technologies (IVF), the IVF associating endochylema, OI is used to carry out nature internal insemination or ins's anovulation patient.
US 4,589,402 with US 4,845,077 the people FSH that does not contain LH of purifying and the application in vitro fertilization thereof are disclosed.EP 322 438 discloses a kind of LH activity that do not have basically, has the active albumen of 6200U/mg FSH at least, and wherein FSH α-subunit and β-subunit possibly be respectively its wild-type or truncation type.
Need long-term treatment in order to obtain result of treatment, usually, need continuous 8-10 days, sometimes up to 21 days, and for the male sex who hangs down gonad-stimulating hormone, induce the spermatogeny need be up to 18 months in order to stimulate generations of women's ovarian follicle.Normally intramuscular injection every day (i.m.) or subcutaneous injection (s.c.) administration of reorganization hFSH, association is uncomfortable and possibly produce the reaction of local injection position.Reduce administration frequency and can promote treatment, make short sexual gland glandular hormone administration more convenient, more be prone to tolerance, more the patient is supreme.
The FSH glycosylation
Gonad-stimulating hormone is gp, and each subunit contains l-asparagine and connects (N-connection) oligosaccharides side chain, and this oligosaccharides side chain is extremely important for activity in vivo and function.It is translation back incident that the sugar of polypeptide adds (glycosylation), and sugar chain is added on l-asparagine (N-connection) or the serine/threonine (O-connection) specifically.Compare with the aminoacid sequence that the protein part of gp is constant, sugared structure is variable, and this specific character is referred to as micro heterogeneity.For example, same proteic each N-glycosylation site can contain different sugared structures.And, even if, also have different sugared structures at a certain proteic same glycosylation site.This heterogeneity is sugared non-template guiding synthetic result.
Proteic N-glycosylation occurs in consistent pattern formula l-asparagine-Xaa-serine/threonine (Asn-Xaa-Ser/Thr) specifically; The less consistent pattern formula l-asparagine-Xaa-halfcystine (Asn-Xaa-Cys) that occurs in, wherein Xaa can be any amino-acid residue.Yet the existence of consistent type tripeptides is not sufficient to guarantee the asparagine residue glycosylation.For example, low 50 times of N-glycosylation rate takes place than other consistent pattern formula Asn-Xaa-Ser/Thr in l-asparagine-proline(Pro)-serine/threonine (Asn-Pro-Ser/Thr) sequence.
People FSH contains 4 N-and connects glycosylation sites: 2 at total α-subunit 52 and 78,2 at β-subunit 7 and 24.The sugar that appends to FSH α-subunit is very crucial for dimeric assembling, integration, secretion and signal transduction, and β-subunit sugar is removed then extremely important from circulation for dimer polymerization, secretion and heterodimer.
People such as Galway, Endocrinology 1990; 127 (1): 93-100 confirms; The FSH two mutants that the Chinese hamster ovary celI system of N-acetylglucosaminyl transferase-I Chinese hamster ovary celI system or sialyl transportation defective produces; Has identical external activity with the hypophysis FSH of wild-type cell excretory or purifying; But but do not have activity in vivo, supposition is because the bad varient of glycosylation is removed very fast in serum.People such as D ' Antonio, Human Reprod1999; 14 (5): 1160-7 has described FSH isoforms (isoform) different in the circulation of blood.These isoforms have identical aminoacid sequence, but the posttranslational modification degree is different.According to finding, to compare with acid isoform, the interior removing of acid more weak isoform body is faster, possibly be because the sialic acid content between isoform is different.In addition, people Endocrinology 1995 such as Bishop; 136 (6): 2635-40 sums up, and circulating half-life is the main determining factor of activity in vivo seemingly.These observations cause a kind of hypothesis, promptly can be through introducing the sialic acid content that extra glycosylation site improves polypeptide, to prolong the transformation period of FSH.
The FSH varient
C-terminal peptide (CTP) through with pregnancy urine extract (hCG) is fused on the natural recombinant human FSH (rhFSH), has developed the FSH agonist the long half-lift of having more.C-terminal peptide (CTP) group is made up of to 145 amino acids 112-118, has to be positioned at 121,127,132 and to be connected glycosylation site with 4 O-of 138.US 5,338, and 835 disclose a kind of FSH β-subunit of modification with US 5,585,345, and its C-terminal glutamate (Glu) prolongs with the CTP group of hCG.It is said that the modification analogue that obtains has the biological activity of natural FSH, but have long circulating half-life.US 5,405, and the C-terminal part of 945 open hCG β-subunits or its varient have material impact for the removing of CG, FSH and LH.
US 5,883,073 disclose have CG, single chain protein TSH, LH and FSH agonist or antagonistic activity and that form by two α-subunits.US 5; 508; 261 disclose the heterodimer polypeptide with LH and fsh receptor binding affinity, and it contains a glycoprotein hormones α-subunit and the β-subunit polypeptide that non-natural produces, wherein; β-subunit polypeptide is to contain 4 to close the amino acid chain of piecing together subsequence, and this subsequence is selected from a group-specific sequence.People such as Klein (2003) disclose FSH analogue a kind of strand, that the transformation period prolongs, α-link to each other through an oligopeptides wherein with β-subunit, and this oligopeptides contains two N-connection glycosylation sites.
For the transformation period in the body that improves FSH, WO 01/58493 discloses 77 kinds of sudden changes can carrying out at α-subunit of FSH and 51 kinds of sudden changes can carrying out at β-subunit of FSH.In addition, WO 01/58493 also discloses and can perhaps insert one or more glycosylation sites in the FSH polypeptide different loci, to improve its transformation period in the terminal increase of the N-of FSH.Can insert the FSH polypeptide though WO 01/58493 has described glycosylation site,, but still can keep the FSH activity not provide guidance for which (which) site to insert glycosylation site in.WO 01/58493 further discloses mutant ' alpha '-and β-subunit (1 extra glycosylation site) or unite use (2 extra glycosylation sites) separately.Certified 128 candidate's two mutants are to use 50 models of three-dimensional (3D) structure of FSH to identify, this model is only based on the structure of hCG and the sequence alignment generation of FSH and hCG, and β-subunit of hCG and FSH has only 32% consistence.WO 01/58493 does not openly introduce any FSH α of glycosylation site-or the production or the mensuration situation of β-subunit through rite-directed mutagenesis.
WO 05/020934 disclose the α of FSH wherein-and β-subunit the GM1 of sudden change is all arranged; Comprise two sudden changes of a β E55N/V57T, promptly L-glutamic acid (E) residue of 55 of amino acid sites Xie Ansuan (V) residue that is mutated into 57 of l-asparagine (N) and amino acid sites is mutated into Threonine (T).The aminoacid sequence of β E55N/V57T is shown in SEQ ID NO:3.
Need a kind of product clinically; The relevant effect of part or all of treatment of FSH can be provided, can more hang down frequency administration than existing FSH product, and preferably; Compare with the accessible circulation FSH activity of existing treatment, more stable activity level can be provided.
The present invention aims to provide the preparation method of this series products and this series products.
Summary of the invention
The FSH molecule that the present invention relates to suddenly change, wherein FSH α-subunit contains sequence SEQ ID NO:4, and FSH β-subunit contains sequence SEQ ID NO:3.The N-glycosylation can take place at 0,1,2,3,4,5 or 6 asparagine residue of described two mutants in this FSH.In one embodiment, the N3 that contains mutant alpha-subunit of SEQ ID NO:4 can be glycosylated.
The invention still further relates to the isolated DNA molecule of a kind of FSH of coding α-subunit two mutants, this α-subunit two mutants contains sequence SEQ ID NO:4.The present invention also relates to a kind of isolated DNA molecule of the FSH of coding β-subunit, this β-subunit contains sequence SEQ ID NO:3.
The invention still further relates to a kind of carrier, this carrier contains the DNA that coding contains FSH α-subunit two mutants of sequence SEQ ID NO:4.This carrier can be an expression vector.
The invention still further relates to a kind of carrier, this carrier contains the DNA that coding contains FSH β-subunit two mutants of sequence SEQ ID NO:3.This carrier can be an expression vector.
The invention still further relates to a kind of carrier, this carrier contains a DNA and the 2nd DNA, and wherein first dna encoding contains FSH α-subunit two mutants of sequence SEQ ID NO:4, and second dna encoding contains FSH β-two mutants of sequence SEQ IDNO:3.This carrier can be an expression vector.
The invention still further relates to a kind of cell, this cell contains the carrier of DNA that coding contains FSH α-subunit two mutants of sequence SEQ ID NO:4.This carrier can be an expression vector.This cell can be a mammalian cell, like Chinese hamster ovary (CHO) cell.
The invention still further relates to a kind of cell, this cell contains the carrier of DNA that coding contains FSH β-subunit two mutants of sequence SEQ ID NO:3.This carrier can be an expression vector.This cell can be a mammalian cell, like Chinese hamster ovary (CHO) cell.
The invention still further relates to a kind of cell; This cell contains the carrier of a DNA and the 2nd DNA; Wherein this first dna encoding contains FSH α-subunit two mutants of sequence SEQ ID NO:4, and second dna encoding contains FSH β-subunit two mutants of sequence SEQ ID NO:3.This carrier can be an expression vector.This cell can be a mammalian cell, like Chinese hamster ovary (CHO) cell.
The invention still further relates to a kind of cell; This cell contains first and second carriers; Wherein first carrier contains the DNA that coding contains FSH α-subunit two mutants of sequence SEQ ID NO:4, and second carrier contains the DNA that coding contains FSH β-subunit two mutants of sequence SEQ IDNO:3.This carrier can be an expression vector.This cell can be a mammalian cell, like Chinese hamster ovary (CHO) cell.
The invention still further relates to a kind of working method of FSH two mutants; Comprise that cultivation can make the mammalian cell of protein glycosylation; Wherein said cell contains first expression vector; This carrier contains the DNA that coding contains FSH α-subunit two mutants of sequence SEQ ID NO:4, and second expression vector, and this carrier contains the DNA that coding contains FSH β-subunit two mutants of sequence SEQ IDNO:3.In yet another embodiment of the present invention; Described cell contains single carrier; This carrier contains the DNA that coding contains FSH α-subunit two mutants of sequence SEQ ID NO:4, also contains the DNA that coding contains FSH β-subunit two mutants of sequence SEQ ID NO:3.
The invention still further relates to a kind of compsn, said composition contains FSH two mutants and pharmaceutically acceptable carrier or vehicle, and wherein FSH α-subunit contains sequence SEQ ID NO:4, and FSH β-subunit contains sequence SEQ ID NO:3.
The invention still further relates to a kind of treatment Mammals sterility method, comprise when needed giving Mammals the FSH two mutants of significant quantity, wherein FSH α-subunit contains sequence SEQ ID NO:4, and FSH β-subunit contains sequence SEQID NO:3.
The invention still further relates to a kind of method that stimulates ovarian follicles to generate; Comprise when needed and give Mammals the FSH two mutants of significant quantity; Wherein FSH α-subunit contains sequence SEQ ID NO:4, and wherein FSH β-subunit contains sequence SEQ ID NO:3.
The invention still further relates to a kind of superovulated method of Mammalian Ovary of inducing; Comprise when needed and give Mammals the FSH two mutants of significant quantity; Wherein FSH α-subunit contains sequence SEQ ID NO:4, and wherein FSH β-subunit contains sequence SEQ ID NO:3.
Description of drawings
The comparison of Fig. 1 α-subunit two mutants D3N (SEQ ID NO:4) and people FSH α-subunit (SEQ ID NO:1).The residue ordinal number refers to people FSH α-subunit (SEQ ID NO:1), wherein first amino acids of 1 its mature polypeptide of finger.
The comparison of Fig. 2 FSH two mutants (this two mutants contains α-subunit two mutants D3N and the β subunit GM1 that contains sequence SEQ ID NO:3) and wild-type FSH amount effect relation curve.Amount effect relation curve has shown that the FSH of different dilution FSH two mutants and wild-type FSH is active.Clone 15C phalangeal process modification D 3N, clone 14C is irrelevant FSH two mutants.
Embodiment
Though show on evidence, the sugared content that improves FSH can cause transformation period prolongation in the body, and it is more more complicated than adding extra glycosylation site simply to improve the FSH transformation period.Though the glycosylation consensus sequence is necessary for adding sugar, is not enough to guarantee that glycosylation site is utilized.Other factors, local protein folding and conformation during biological example is synthetic are determining whether oligosaccharides can add specific consensus sequence to.In addition, in order to reach the purpose that transformation period in the body improves with extra glycosylation, consensus sequence must be in such position, i.e. the glycosylation in this site can not disturbed receptors bind or jeopardize folding, the conformation or the stability of this gp.In this, have the FSH analogue of longer transformation period, mainly be defined in fusion rotein, its polypeptide merges part and comprises extra glycosylation site.
The FSH two mutants
The invention provides a kind of FSH mutant, can produce extra glycosylation recognition site through modifying.Compare with the α-subunit of wild-type, the α-subunit of FSH two mutants can be a kind of in the following sudden change: D3N and Q5T.The FSH two mutants can contain above-mentioned α-subunit two mutants and β-subunit two mutants, and for example β GM1 contains following sudden change: E55N/V57T.The one or more extra glycosylation site of RECFSH can be glycosylated.These one or more extra glycosylation sites can be in external glycosylation or glycosylation in vivo.
The FSH two mutants can be used the known method preparation of any suitable prior art.These methods comprise the nucleotide sequence of the corresponding FSH two mutants of structure coding and express amino acid sequence in the transfecting host that is fit to.The FSH two mutants can also adopt the chemosynthesis preparation, perhaps uses chemosynthesis and recombinant DNA technology to prepare through uniting.
The FSH two mutants can contain the FSH α that exists with two independent polypeptied chain forms-and β-subunit; These two chains in vivo dimerization to produce the dimer polypeptide; Perhaps it can contain the strand construct, and this construct contains through peptide bond or covalently bound two subunits of connection peptides.The amino-acid residue of connection peptides has and that it(?) can not can obviously disturbs the active character of FSH two mutants.
The FSH two mutants is compared with wild-type FSH, can have the longer transformation period.FSH compares with wild-type, and the FSH two mutants can also have better stability.The FSH two mutants can connect glycosylation site 0,1,2,3,4,5 or 6 at N-and contain oligosaccharides.The present invention also provides one group of FSH two mutants, and they can contain one or more isoform FSH two mutants, and wherein every kind of isoform contains at N-connection glycosylation site O, 1,2,3,4,5 or 6 and contains oligosaccharides.
Coding FSH mutant alpha-or β-subunit nucleotide sequence; For example have the hFSH-α or the hFSH-β of the aminoacid sequence shown in SEQ ID NOS:1 and 2 respectively; Can make up through separating, or make up through the nucleotide sequence of composite coding FSH subunit fundamental chain.Can change replacement or the insertion of nucleotide sequence then to realize corresponding amino-acid residue.Nucleotide sequence can be modified through rite-directed mutagenesis.Perhaps, nucleotide sequence can also be through chemosynthesis preparation, and wherein oligonucleotide is according to the special aminoacid sequence design of FSH two mutants.
The nucleotide sequence of coded polypeptide can be inserted into recombinant vectors, and operability be connected to regulating and controlling sequence, this regulating and controlling sequence is that the target host cell express polypeptide of transfection is necessary.Regulating and controlling sequence can be any for the essential or useful component of expression of polypeptides.The regulating and controlling sequence of example as instructing mammalian cell to transcribe of the regulating and controlling sequence that is fit to; Comprise the early stage and late promoter of SV40 and adenovirus; Like adenovirus-2 major late promoter, MT-1 (metallothionein gene) promotor and human cytomegalic inclusion disease virus (CMV) be early promoter at once.
The technician in present technique field does not need undue experimentation just can in these carriers, expression regulation sequence and host cell, select.Recombinant vectors can be an autonomously replicationg vector, the carrier that promptly exists with the outer entity of karyomit(e), and it duplicates and is independent of chromosome duplication, for example plasmid.Perhaps, carrier can also be when it introduces host cell, the carrier that is incorporated into the host cell gene group and duplicates jointly with one or more karyomit(e)s that it is integrated.
Carrier can be an expression vector, and the nucleotide sequence of the polypeptide of the present invention of wherein encoding can be connected to operability other Nucleotide and transcribe on the needed fragment.Carrier can derive from plasmid or viral DNA.Mention that here a large amount of carriers that is adapted at host cell expression is commercially available or is shown in document description.
Recombinant vectors can further include the dna sequence dna that carrier is duplicated in described host cell.The example of such sequence, (when host cell is mammalian cell) are the SV40 replication orgin.Carrier can also contain selective marker; For example its product and host's defective complementary gene; As the gene of the Tetrahydrofolate dehydrogenase of encoding (DHFR); Perhaps express the gene of drug resistance, like penbritin, kantlex, tsiklomitsin, paraxin, Xin Meisu, Totomycin or methotrexate resistant gene.
Carrier can also comprise amplifiable gene such as DHFR, so that contain the amplifiable gene of a plurality of copies and the cell of flanking sequence, comprises the cell of FSH two mutants DNA, can on suitable substratum, be screened.
The present invention also provides the DNA of coding FSH mutant alpha-subunit.No matter the nucleotide sequence of this coding FSH mutant alpha-subunit and β subunit is to prepare through rite-directed mutagenesis, synthetic, PCR or other method, can also randomly comprise the nucleotide sequence of coded signal peptide.Need be from the cell of expressing it excretory the time when polypeptide, can there be this signal peptide.The sort signal peptide if exist, can be selected the cell recognition as expression of polypeptides.Signal peptide can be homologous (as normally related with the hFSH subunit) with polypeptide; It also can be allogenic (promptly coming from the another kind source outside the hFSH); Can be homologous or allogenic perhaps with host cell; Promptly usually at the signal peptide of this host cell expression, or usually not at the signal peptide of this host cell expression.
Any suitable host can be used for producing this polypeptide, comprises bacterium, fungi (comprising yeast), plant, insect, Mammals, or other zooblast or animal cell line that is fit to, and transgenic animal or plant.The example of the mammalian host cell line that is fit to comprises Chinese hamster ovary (CHO) clone, (like CHO-KL; ATCC CCL-61), grivet clone (COS) (like COS 1 (ATCC CRL-1650), COS 7 (ATCCCRL-1651)); Mouse cell lines (like NSIO), young hamster kidney (BI-EK) clone (like ATCC CRL-1632 or ATCC CCL-10), and human cell line (like BEK 293 (ATCC CRL-1573)), and the vegetable cell of tissue culture.Other the clone that is fit to is known for the present technique field, can collect center (American Type Culture Collection, USA) acquisition from public preservation mechanism such as US mode bacterial classification.The method that foreign DNA is introduced mammalian host cell comprises the transfection, electroporation, the transfection of diethylin ethyl-DEXTRAN 500.000 (DEAE-dextran) mediation, liposome-mediated transfection and virus vector of calcium phosphate mediation.
Can adopt prior art, culturing cell in the nutritional medium that is fit to polypeptide production.For example; Can be through shake-flask culture, small-scale or fairly large fermentation (comprise continuously, batch, stream adds or solid state fermentation) culturing cell; Can in laboratory or industrial fermentation jar, carry out under expression of polypeptides and/or the isolating condition with allowing at the substratum that is fit to.Cultivation adopts the known program of prior art to carry out in the nutritional medium that contains carbon source, nitrogenous source and inorganic salt.The substratum that is fit to can be provided by commercial supplier, perhaps according to disclosed component preparation (collecting the catalogue at center like the US mode bacterial classification).If polypeptide is secreted in the nutritional medium, can directly from substratum, reclaim.If polypeptide is an excretory not, can from cell lysate, reclaim.The method of a High-efficient Production FSH two mutants of the present invention is through using dihydrofolate dehydrogenase (DHFR) in DHFR-defective type Chinese hamster ovary celI, to increase, realizing that through the concentration that increases continuously methotrexate like United States Patent(USP) No. 4,889,803 is said.
The FSH mutant polypeptide that produces can use the known method of prior art to reclaim.For example, can use traditional method from nutritional medium, to reclaim, include but not limited to centrifugal, filtration, extraction, spraying drying, evaporation or deposition.The FSH mutant polypeptide can be used the known method purifying of multiple prior art, includes but not limited to chromatography (like IX, affine, hydrophobic, chromatofocusing and size-exclusion), electrophoresis method (like point focusing such as preparation types), difference solubleness (like ammonium sulphate precipitation), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or extraction.
The present invention also provides a kind of pharmaceutical composition of the FSH of containing two mutants.This pharmaceutical composition can be used to stimulate ovarian follicle to generate, and for example unites use with ovulation induction or auxiliary procreation technology (ART).Because FSH two mutants of the present invention can effectively be induced many follicular developments and maturation, it possibly be particularly suitable for expecting to collect the ART of a plurality of ovum.
Induce single ovarian follicle to generate when the FSH two mutants can be used to bring out ovulation (OI), perhaps a small amount of ovarian follicle during ins (IUI) generates (paucifolliculogenesis reaches about 3 ovarian follicles), is used for internal insemination.Than traditional F SH preparation, the FSH two mutants reduces consumption or the low frequency medication can obtain single ovarian follicle generation.For example, in bringing out ovulation (OI), FSH preparation of the present invention can give 225-400IU in per 3 days, perhaps low dosage more, and this depends on reaction.Patient's reaction can use ultrasonic figure to follow the tracks of.
FSH two mutants of the present invention can be used for controlled ultra ovulation (COH) scheme.The COH standard scheme comprises the downward modulation phase; In this phase,, be stimulation period through giving gonadotropin releasing hormone (GnRH) agonist downward modulation endogenous lutropin (LH) thereupon; In this phase; Through giving follicular stimulating hormone (FSH) every day, about 150-225IU/ days usually, induce follicular development (ovarian follicle generation).Perhaps, stimulation can begin with FSH after spontaneous or inductive menstruation, gives GnRH antagonist (usually from stimulation period front and back beginning in the 6th day) subsequently.When at least 3 ovarian follicles＞16 millimeter (one is 18mm), give single dose inject hCG (5-10,000IU), simulating nature LH peak (LH surge), induced ovulation.The ovum results were regularly injected the back 36-38 hour at hCG.
This FSH two mutants can also be used for OI and IUI.For example, can after spontaneous or inductive menstruation, begin FSH stimulates, and every day, dosage was 75-150 IU.When 1 or 3 FD reaches at least 16 millimeters, give single dose and inject hCG induced ovulation.Insemination can be through carrying out in conventional sexual intercourse or the IUI body.
Because the FSH two mutants can have the longer transformation period than wild-type FSH preparation; Can use the FSH preparation of low IU dosage in the above-mentioned scheme; And/or can come the adjustment scheme through shortening FSH thorn flyback cycle, and the quantity of ovarian follicle and active aspect, can reach same or better reaction.For example, every day dosage for or the FSH that is approximately 50-150,50-100 or 50-75IU just can realize enough ovarian follicle generations.FSH dosage can be to be every day or half day the basis.The administration cycle can be less than or be approximately 14,12,11 or 10 days.For OI, the FSH mutant preparation can be 25-150 or 50-125IU FSH/ days administration.For the treatment of male sterility, the FSH mutant preparation can reach the level that is enough to through conventional sexual intercourse or the fertilization of ART technology according to 3X150～300IU/ week administration until spermatogeny.
Because the FSH two mutants has the long transformation period than wild-type FSH preparation, can be used as the prolonged action preparation administration, said preparation can be to be lower than frequency administration once in per two days.Traditional F SH can administration in per two days, and consumption is or about 300IU, perhaps administration every day, and consumption be or about 150IU that effect is similar.The FSH two mutants can administration in per 3,4,5,6 or 7 days, and reaches the similar or better effect with traditional F SH.
The FSH two mutants can be used to produce the medicine that is used for treating disease, disorder or symptom.On the other hand, polypeptide of the present invention or pharmaceutical composition can be used on the treatment Mammals, in the particularly human method, comprise the step that gives this polypeptide of Mammals or pharmaceutical composition when needed.
Technician for the present technique field; Obviously; The significant quantity of one peptide species, preparation or compsn; No matter this polypeptide, preparation or compsn be independent medication or and other medicine unite use, depend on disease, dosage, administration time table, said composition serum half-life and patient the general health situation how.Usually, the effective dose of said preparation or compsn is enough to guarantee result of treatment.
The FSH two mutants can be with the compsn administration, and said composition comprises one or more pharmaceutically acceptable carriers or vehicle." pharmacy is acceptable " is meant that a kind of carrier or vehicle can not make the patient who is given this medicine produce any untoward reaction.This pharmaceutically acceptable carrier and vehicle are well-known in the present technique field; This polypeptide can be processed pharmaceutical composition (referring to book of reference according to well-known method; As: Remington ' sPharmaceutical Sciences, 18th edition, A.R.Gennaro; Ed., Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaerand L.Hovgaard, Eds., Taylor & Francis (2000); With Handbook of PharmaceuticalExcipients, 3rd edition, A.Kibbe, Ed., Pharmaceutical Press (2000)).The acceptable vehicle of pharmacy that can be used for containing the compsn of this polypeptide comprises; For example, buffer reagent, stablizer, sanitas, isotonic regulator (isotonifiers), nonionogenic tenside or stain remover (" wetting agent "), inhibitor, swelling agent or weighting agent, sequestrant and solubility promoter.
The pharmaceutical composition that contains the FSH two mutants can be processed various formulations, comprise liquid as promptly with solution or suspension-s, gel, freeze dried, perhaps any formulation that other is fit to, as be fit to process the dry powder or the crystal of solution.The form of compsn can depend on the special indication of needs treatment, and this is conspicuous for those skilled in the art.
This contain the FSH two mutants pharmaceutical composition can through vein, through muscle, through peritonaeum, through skin, through subcutaneous, through the hypogloeeis, trans-oral, via intranasal application, transdermal administration; Or through sucking and any other acceptable manner such as dry powder injection (PowderJect) or a ProLease encapsulation technology or an injecting systems; Mode of administration will depend on the special indication of needs treatment, and this is conspicuous for those skilled in the art.Said composition can be through subcutaneous administration, and patient can carry out self-medication like this.
This pharmaceutical composition can be united use with other medicine.These medicines may be mixed in in this pharmaceutical composition as its part, also can separate with polypeptide, and administration is simultaneously perhaps used according to other acceptable regimen.In addition, this polypeptide, preparation or pharmaceutical composition can be done the auxiliary use of other treatment.
The present invention includes many aspects, specify by following nonrestrictive example.
The FSH two mutants
The 3D crystalline structure of people FSH once was used for discerning the position that can insert candidate's glycosylation site on candidate's glycosylation site on FSH α-subunit or the FSH molecule.In the asymmetric group of each crystalline structure, there are two FSH molecules (four subunits).These two FSH molecules are superposeed and compare, and each residue visually all can be observed, and to discern potential site, this site can be inserted the N-glycosylation site and can not disturbed the correct folding of FSH polypeptide or reduce FSH active.The crystal collection of illustrative plates structure of FSH and combine about the knowledge of FSH/FSHR acceptor interaction helps further to select potential N-glycosylation site.Main standard is exactly that the 3D structure deteriorate combination that minimizes, predict and avtive spot destroy and minimize, and the 3D structure and the glycosylation of prediction are compatible.Based on above standard, identify the two mutants of following FSH α-subunit aminoacid sequence:
| # | Sudden change | SEQ?ID?NO |
| 1 | D3 | 4 |
Embodiment 2
The morphological analysis of FSH two mutants
Get spissated culture supernatants from the FSH α-subunit two mutants of transient expression and be divided into some equal portions, under non-reduced condition, carry out SDS-PAGE and analyze, this condition can allow not to be split into free α-and β-subunit by complete FSH dimer.Through comparing the apparent molecular weight of each two mutants dimer and wild-type FSH, can confirm whether two mutants FSH compares with wild-type FSH is ultra glycosylated.In brief, behind the electrophoresis, albumen to PVDF (PVDF) film, is used Xue Lannuo (Serono) the antibody 9-14 of anti-FSH α-subunit to develop by electrophoretic transfer.As contrast, wild-type FSH, two mutants GM1, FSH-C-terminal peptide (FSH-CTP) and the sweet smell (Gonal F) of really receiving are also analyzed.Table 1 shows the apparent molecular weight based on the heterodimer of being made up of α-subunit two mutants and wild-type beta-subunit of molecular weight standard article calculating.
Table 1
| Sample | Relative molecular weight (kDa) | Cycles of concentration |
| ?FSH-CTP | 45.244 | |
| Fruit receives sweet smell | 38.482 | 37x |
| ?D3N | 46.158 | 39x |
| ?GM1 | 46.202 | 40x |
| Wild-type FSH | 45.083 | 36x |
FSH two mutants as shown in table 1, expressed, promptly the D3 two mutants shows that level of glycosylation increases, and evidence is to compare with wild-type people FSH, and this heterodimer apparent molecular weight is distributed with variation.
Embodiment 3
The external function of FSH two mutants
In order to measure the activity of FSH two mutants, having measured each two mutants stimulates the Chinese hamster ovary celI of recombinant expressed people's fsh receptor to produce the ability of cyclic monophosphate (cAMP).CHO-FSH acceptor (CHO-FSHR) cell remains on [minimum medium (MEM) (-) (Gibco in the FSHR growth medium; Cat#12561-056)+10% dialysis-type foetal calf serum (Gibco; Cat#26300-020)+600 μ g/ml microbiotic Geneticin (Gibco, cat#10131-035)+0.02 μ M methotrexate].The CHO-FSHR cell is with 2 * 10
4The cells/well inoculation, 100 μ l/ holes (2 * 10
6Cell/10ml=1 ware), before detection, hatched 24 hours in 37 ℃.At least 70% cell when converging is used for measuring.
All samples and interior mark (Gonal F is as interior mark) begin to carry out 1: 3 serial dilution of 12-point from 67.5nM.All [DMEM/F12 (does not contain phenol red detecting substratum in all dilutions; Gibco, cat#11039-021)+the 1mg/ml foetal calf serum (Sigma, A-6003)+0.1mM IBMX (3-isobutyl--1-methyl xanthone-phosphodiesterase inhibitor; Carry out Sigma, cat#I-7018)].From detect ware, remove growth medium, add 25 μ l and detect substratum (with MA6000 cAMP MSD test kit-Meso Scale Discovery, Gaithersburg, MD provides), reclaim petridish and hatched 15 minutes at 37 ℃.Add 25 μ l/ hole testing samples in the hole, mix, reclaim petridish and hatched 1 hour at 37 ℃.After hatching 1 hour, from the hole, remove sample and substratum.Every then hole adds 25 μ l standard lysis buffers (providing with MA 6000 Meso Scale Discovery test kits), and petridish adds a cover that (Packard cat#6005185) sways 5 minutes.After cracking in 5 minutes is hatched, 25 μ l cell lysates are transferred in the cAMP Meso Scale Discovery petridish (providing with the MA6000MSD test kit) the gentle mixed culture of room temperature 30 minutes.Every hole adds 25 μ l cAMP-alkaline phosphatase enzyme composition, and every then hole adds the anti-cAMP antibody of 25 μ l, and petridish is added a cover room temperature and swayed 30 minutes.Petridish then cleans 6 times with automatic rinser with 350 μ l/ hole cleaning buffer solutions.Every then hole adds 100 μ Sapphire II RTU (instant) substrate reinforcing agents, and petridish is added a cover 25 ℃ of dark places and hatched 30 minutes.The 1 second reading in the every hole of petridish hangs down cAMP concentration person and shows high signal then, and high cAMP concentration person shows low signal.The amount effect relation curve of FSH two mutants is as shown in Figure 2.Calculate medium effective concentration (EC50) value, see table 2.
Shown in Fig. 2 and table 2, the FSH two mutants has the external activity suitable with wild-type FSH.
Table 2
| Sample | EC 50M |
| Wild-type FSH | 1.99E-10 |
| Clone 15C-α D3N/ β GM1 | 4.14E-10 |
Embodiment 4
Transformation period in the body of FSH two mutants
The D3N two mutants of two different batches uses independently pharmacokinetic to analyze.Same design is taked in two researchs: the prematurity female sd inbred rats of 33 21 ages in days (the about 40g of body weight; Charles RiverLaboratories, Wilmington MA) is divided into 5 treatment groups (n=6) and a baseline group (n=3) at random.Select the prematurity female rats to be based on and to use this age and sex to carry out Determination of biological activity in the body of FSH.The treatment treated animal is accepted subcutaneous (s.c.) injection 4ug GM1 (contrast), D3N two mutants or 8ug Gonal-F (rFSH) and is really received fragrant people's RECFSH (Gonal-F (rFSH)) (contrast).0 hour from the baseline treated animal, gets blood (n=3/ time point from treatment treated animal hole behind ball in 1,2,4,6,10,24,48 and 72 hour; Rat is got blood in turn so that two follow-up sample spot can be not hemorrhage).Blood 0.1ml is approximately got in every each blutpunkte of rat, and results blood plasma is stored in-80 ℃ and treats elisa assay.In two research in the used detection serum the proteic method of FSH be with DSL FSH encapsulate orifice plate ELISA (Diagnostics Systems Laboratories, Webster, TX).Each serum sample replicate analysis three times.
The transformation period of D3N two mutants is apparently higher than wild-type FSH.
Embodiment 5
BA in the body
The model of estimating BA in the D3N two mutants body is the rat ovary added weight method.Use FSH or have the similar active molecule of FSH,, treat immature 21 age in days female rats, trigger folliculus ovarii growth and ovum and produce like the D3N two mutants.This growth is easy to be detected through measuring treatment ovary weight in latter stage.In this model, material to be detected passed through drug administration by injection 3 days, took out ovary the last time after the administration and weighed.This method has been used many decades as the basis of the purpose of giving the active assignment of clinical prods label F SH.The related physiological effect of its energy measurement FSH has clear and definite dependency with the performance of clinical prods.
The activity in vivo of D3N two mutants and wild-type FSH is compared.Based on the equivalence of the FSH that predicts, and consider its external activity and, define all dosage in the intravital transformation period of rat.Find that the D3N two mutants has very strong FSH activity, its intensity is similar with wild-type FSH.
Claims (15)
1. FSH two mutants, wherein β-subunit contains sequence SEQ ID NO:3, and wherein α-subunit contains sequence SEQ ID NO:4.
2. FSH two mutants as claimed in claim 1, wherein any one is glycosylated in the asparagine residue of 0-6 position.
3. FSH two mutants as claimed in claim 1, wherein the N3 of α-subunit is glycosylated.
4. carrier, it comprises the nucleic acid of encoding amino acid sequence SEQ ID NO:3 and the nucleic acid of encoding amino acid sequence SEQ ID NO:4.
5. carrier as claimed in claim 4, wherein this carrier is an expression vector.
6. host cell that contains the described carrier of claim 4.
7. host cell as claimed in claim 6, wherein said cell is a mammalian cell.
8. the working method of the described FHS two mutants of claim 1 comprises:
(a) a kind of cell is provided, this cell comprises first nucleic acid of encoding amino acid sequence SEQ ID NO:3 and second nucleic acid of encoding amino acid sequence SEQ ID NO:4;
(b) under the condition that allows first and second expression of nucleic acid, cultivate this cell.
9. method as claimed in claim 8, wherein said cell can make protein glycosylation.
10. method as claimed in claim 9, wherein this cell contains single carrier, and this carrier contains the nucleic acid of encoding amino acid sequence SEQ ID NO:3 and the nucleic acid of encoding amino acid sequence SEQ ID NO:4.
11. method as claimed in claim 9, wherein this cell contains: first carrier and second carrier that (b) contains the nucleic acid of encoding amino acid sequence SEQ ID NO:4 that (a) comprise the nucleic acid of encoding amino acid sequence SEQ ID NO:3.
12. a pharmaceutical composition, said composition contain the described FSH two mutants of claim 1, randomly also contain pharmaceutically acceptable carrier or vehicle.
13. the application of FSH two mutants as claimed in claim 1 in the sterile medicine of preparation treatment Mammals.
14. the application of FSH two mutants as claimed in claim 1 in the medicine that preparation stimulation ovarian follicles generates.
15. FSH two mutants as claimed in claim 1 is induced the application in the superovulated medicine of Mammalian Ovary in preparation.
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| US75948606P | 2006-01-17 | 2006-01-17 | |
| US60/759,486 | 2006-01-17 | ||
| PCT/US2007/001004 WO2007084441A2 (en) | 2006-01-17 | 2007-01-16 | Novel fsh glycosylation variant d3n |
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| US8604175B2 (en) * | 2005-12-09 | 2013-12-10 | Ares Trading S.A. | Method for purifying FSH or a FSH mutant |
| WO2008010840A2 (en) * | 2005-12-22 | 2008-01-24 | Laboratoires Serono S.A. | Fsh mutants |
| CA2702448A1 (en) * | 2007-10-22 | 2009-04-30 | Merck Serono S.A. | Method for purifying fc-fusion proteins |
| EP2203465A1 (en) * | 2007-10-22 | 2010-07-07 | Merck Serono S.A. | Method for purifying an fc-containing protein |
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| WO2001058493A1 (en) | 2000-02-11 | 2001-08-16 | Maxygen Aps | Conjugates of follicle stimulating hormones |
| ES2389835T3 (en) * | 2001-10-22 | 2012-11-02 | Merck Serono Sa | Mutant glycoproteins |
| UA88879C2 (en) * | 2003-09-02 | 2009-12-10 | Эплайд Рисерч Системз Эрс Холдинг Н.В. | Fsh glycosylation mutant |
| US8604175B2 (en) * | 2005-12-09 | 2013-12-10 | Ares Trading S.A. | Method for purifying FSH or a FSH mutant |
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| PT1981908E (en) | 2010-11-23 |
| DK1981908T3 (en) | 2010-12-13 |
| CA2633844A1 (en) | 2007-07-26 |
| DE602007010425D1 (en) | 2010-12-23 |
| WO2007084441A2 (en) | 2007-07-26 |
| AU2007207697A1 (en) | 2007-07-26 |
| US7956034B2 (en) | 2011-06-07 |
| BRPI0706628A2 (en) | 2011-04-05 |
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| SI1981908T1 (en) | 2011-01-31 |
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